Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells
[EN] Accurate detection of H. pylori in different environmental and clinical samples is essential for public health studies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete...
| Autores: | , , , |
|---|---|
| Tipo de recurso: | artículo |
| Fecha de publicación: | 2021 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | inglés |
| OAI Identifier: | oai:riunet.upv.es:10251/189668 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/189668 |
| Access Level: | acceso abierto |
| Palabra clave: | Helicobacter pylori PMA-q PCR PEMAX(TM)-qPCR Viability Disinfection treatment Morphological states MICROBIOLOGIA |
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| dc.title.none.fl_str_mv |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| title |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| spellingShingle |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells Hortelano, Irene Helicobacter pylori PMA-q PCR PEMAX(TM)-qPCR Viability Disinfection treatment Morphological states MICROBIOLOGIA |
| title_short |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| title_full |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| title_fullStr |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| title_full_unstemmed |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| title_sort |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells |
| dc.creator.none.fl_str_mv |
Hortelano, Irene Moreno Trigos, Mª Yolanda|||0000-0003-3688-5157 García Hernández, Jorge|||0000-0003-1258-6128 Ferrús Pérez, Mª Antonia |
| author |
Hortelano, Irene |
| author_facet |
Hortelano, Irene Moreno Trigos, Mª Yolanda|||0000-0003-3688-5157 García Hernández, Jorge|||0000-0003-1258-6128 Ferrús Pérez, Mª Antonia |
| author_role |
author |
| author2 |
Moreno Trigos, Mª Yolanda|||0000-0003-3688-5157 García Hernández, Jorge|||0000-0003-1258-6128 Ferrús Pérez, Mª Antonia |
| author2_role |
author author author |
| dc.contributor.none.fl_str_mv |
Departamento de Biotecnología Instituto Universitario de Ingeniería del Agua y del Medio Ambiente Centro Avanzado de Microbiología Aplicada Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural Generalitat Valenciana Agencia Estatal de Investigación Ministerio de Economía y Competitividad Repositorio Institucional de la Universitat Politècnica de València Riunet |
| dc.subject.none.fl_str_mv |
Helicobacter pylori PMA-q PCR PEMAX(TM)-qPCR Viability Disinfection treatment Morphological states MICROBIOLOGIA |
| topic |
Helicobacter pylori PMA-q PCR PEMAX(TM)-qPCR Viability Disinfection treatment Morphological states MICROBIOLOGIA |
| description |
[EN] Accurate detection of H. pylori in different environmental and clinical samples is essential for public health studies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete exclusion of dead cells amplification signals. The use of DNA intercalating dyes has been proposed. However, its efficacy is still not well determined. In this study, we aimed to test the suitability of PMA and PEMAXTM dyes used prior to qPCR for only detecting viable cells of H. pylori. Their efficiency was evaluated with cells submitted to different disinfection treatments and confirmed by the absence of growth on culture media and by LIVE/DEAD counts. Our results indicated that an incubation period of 5 min for both, PMA and PEMAXTM, did not affect viable cells. Our study also demonstrated that results obtained by using intercalating dyes may vary depending on the cell stress conditions. In all dead cell¿s samples, both PMA and PEMAXTM pre-qPCR treatments decreased the amplification signal (>103 Genomic Units (GU)), although none of them allowed for its disappearance confirming that intercalating dyes, although useful for screening purposes, cannot be considered as universal viability markers. To investigate the applicability of the method specifically to detect H. pylori cells in environmental samples, PMA-qPCR was performed on samples containing the different morphological and viability states that H. pylori can acquire in environment. The optimized PMA-qPCR methodology showed to be useful to detect mostly (but not only) viable forms, regardless the morphological state of the cell. |
| publishDate |
2021 |
| dc.date.none.fl_str_mv |
2021 2021-06-01 |
| dc.type.none.fl_str_mv |
journal article http://purl.org/coar/resource_type/c_6501 VoR http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
https://riunet.upv.es/handle/10251/189668 |
| url |
https://riunet.upv.es/handle/10251/189668 |
| dc.language.none.fl_str_mv |
Inglés eng |
| language_invalid_str_mv |
Inglés |
| language |
eng |
| dc.relation.none.fl_str_mv |
Agencia Estatal de Investigación http://dx.doi.org/10.13039/501100011033 Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020 PID2019-105691RB-I00 ANALISIS INTEGRADO DE LA DIVERSIDAD, CALIDAD Y SEGURIDAD MICROBIOLOGICA DE ALIMENTOS VEGETALES ECOLOGICOS MEDIANTE CULTIVO, TECNICAS MOLECULARES Y SECUENCIACION MASIVA Generalitat Valenciana https://doi.org/10.13039/501100003359 AICO%2F2018%2F273 DETERMINACION DEL RIESGO PARA LA SALUD PUBLICA DEBIDO A LA PRESENCIA DE H. PYLORI EN AGUA Y ALIMENTOS: DETECCION Ministerio de Economía y Competitividad http://dx.doi.org/10.13039/501100003329 AGL2014-53875-R H. PYLORI Y OTROS HELICOBACTERS PATOGENOS EN AGUAS Y ALIMENTOS: DESARROLLO Y APLICACION DE HERRAMIENTAS MOLECULARES DIRIGIDAS A LA EVALUACION DEL RIESGO PARA EL CONSUMIDOR |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Reconocimiento - No comercial - Sin obra derivada (by-nc-nd) http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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info:eu-repo/semantics/openAccess |
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open access http://purl.org/coar/access_right/c_abf2 Reconocimiento - No comercial - Sin obra derivada (by-nc-nd) http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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openAccess |
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application/pdf application/pdf |
| dc.publisher.none.fl_str_mv |
Elsevier |
| publisher.none.fl_str_mv |
Elsevier |
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reponame:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia instname:Universitat Politècnica de València (UPV) |
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Universitat Politècnica de València (UPV) |
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RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
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RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
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1869410575805579264 |
| spelling |
Optimization of pre- treatments with Propidium Monoazide and PEMAX¿ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cellsHortelano, IreneMoreno Trigos, Mª Yolanda|||0000-0003-3688-5157García Hernández, Jorge|||0000-0003-1258-6128Ferrús Pérez, Mª AntoniaHelicobacter pyloriPMA-q PCRPEMAX(TM)-qPCRViabilityDisinfection treatmentMorphological statesMICROBIOLOGIA[EN] Accurate detection of H. pylori in different environmental and clinical samples is essential for public health studies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete exclusion of dead cells amplification signals. The use of DNA intercalating dyes has been proposed. However, its efficacy is still not well determined. In this study, we aimed to test the suitability of PMA and PEMAXTM dyes used prior to qPCR for only detecting viable cells of H. pylori. Their efficiency was evaluated with cells submitted to different disinfection treatments and confirmed by the absence of growth on culture media and by LIVE/DEAD counts. Our results indicated that an incubation period of 5 min for both, PMA and PEMAXTM, did not affect viable cells. Our study also demonstrated that results obtained by using intercalating dyes may vary depending on the cell stress conditions. In all dead cell¿s samples, both PMA and PEMAXTM pre-qPCR treatments decreased the amplification signal (>103 Genomic Units (GU)), although none of them allowed for its disappearance confirming that intercalating dyes, although useful for screening purposes, cannot be considered as universal viability markers. To investigate the applicability of the method specifically to detect H. pylori cells in environmental samples, PMA-qPCR was performed on samples containing the different morphological and viability states that H. pylori can acquire in environment. The optimized PMA-qPCR methodology showed to be useful to detect mostly (but not only) viable forms, regardless the morphological state of the cell.This work was supported by the Conselleria de Educacion, Investigacion, Cultura y Deporte, of the Community of Valencia, Spain, under project AICO/2018/273, and by the Spanish Ministerio de Ciencia e Innovacion PID2019-105691RB-I00 Grant and by the Spanish Ministry of Economy and Competitiveness AGL2014/53875-R Grant.ElsevierDepartamento de BiotecnologíaInstituto Universitario de Ingeniería del Agua y del Medio AmbienteCentro Avanzado de Microbiología AplicadaEscuela Técnica Superior de Ingeniería Agronómica y del Medio NaturalGeneralitat ValencianaAgencia Estatal de InvestigaciónMinisterio de Economía y CompetitividadRepositorio Institucional de la Universitat Politècnica de València Riunet20212021-06-01journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfapplication/pdfhttps://riunet.upv.es/handle/10251/189668reponame:RiuNet. Repositorio Institucional de la Universitat Politécnica de Valénciainstname:Universitat Politècnica de València (UPV)InglésengAgencia Estatal de Investigación http://dx.doi.org/10.13039/501100011033 Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020 PID2019-105691RB-I00 ANALISIS INTEGRADO DE LA DIVERSIDAD, CALIDAD Y SEGURIDAD MICROBIOLOGICA DE ALIMENTOS VEGETALES ECOLOGICOS MEDIANTE CULTIVO, TECNICAS MOLECULARES Y SECUENCIACION MASIVAGeneralitat Valenciana https://doi.org/10.13039/501100003359 AICO%2F2018%2F273 DETERMINACION DEL RIESGO PARA LA SALUD PUBLICA DEBIDO A LA PRESENCIA DE H. PYLORI EN AGUA Y ALIMENTOS: DETECCIONMinisterio de Economía y Competitividad http://dx.doi.org/10.13039/501100003329 AGL2014-53875-R H. PYLORI Y OTROS HELICOBACTERS PATOGENOS EN AGUAS Y ALIMENTOS: DESARROLLO Y APLICACION DE HERRAMIENTAS MOLECULARES DIRIGIDAS A LA EVALUACION DEL RIESGO PARA EL CONSUMIDORopen accesshttp://purl.org/coar/access_right/c_abf2Reconocimiento - No comercial - Sin obra derivada (by-nc-nd) http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:riunet.upv.es:10251/1896682026-06-13T07:49:27Z |
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15.301603 |